Skip to main content

Chemical Biology

21st Century Enzymes

Here’s a remarkable paper that shows how enzymes can be engineered to turn out some very unnatural-looking structures. Frances Arnold’s group at CalTech has published on a great deal of work in this area in the past, and this latest variation is something to see. They’ve been working with various heme proteins, since that reactive center is known to be able to generate carbenes, and they noted one variant, in the presence of ethyl diazoacetate as a carbene source and an aryl acetylene as reactant, gave furan products.

These are already reported as coming from a carbene mechanism, so the group thought that they must be on to something. Further screening of variants around that particular enzyme isoform turned up some that allowed a second carbene reaction to cycle around, giving the unusual bicyclobutanes shown at right. Freeing up some room around the active site was essential, otherwise the cyclopropene intermediate goes off to the furan product instead. A systematic screen of amino acid variations at the key residues of the enzyme in E. coli, with monitoring of product production, furnished an enzyme that was capable of high turnover (enough to produce hundreds of milligrams of product). A variety of different R groups are tolerated on the phenyl, with yields from 30 to 80%.

In this case, the phenylcyclopropene intermediate was not isolable, but the team also look for variants that could produce those products as well (in the non-aryl cases). Similar mutation screens against promising enzymes produced another one that could indeed furnish a variety of cyclopropenes, as shown at right, in yields of 35 to 85%. Variants were found that could produced enhanced ratios of either enantiomer, to boot.

These sorts of reactions are known in the literature, up to a point (here’s a review presentation from the Wipf group a few years back on the topic). But such things are mostly seen on very electron-deficient alkynes and often go in lower yields than what are reported here (and I think you can largely forget about enantioselectivity). The fact that enzymes can be made to do such chemistry is remarkable, and there are surely many more such things waiting to be discovered. The metal centers in these proteins do a lot of extraordinary chemistry, and many groups are in the process of harnessing them for synthetic purposes. Seeing unusual strained systems like this being formed so readily just shows how much must be possible!

36 comments on “21st Century Enzymes”

  1. Marcus Theory says:

    Did Arnold move to MIT? I thought she was at CalTech. I don’t keep up with all the professor moves anymore so maybe….

    1. Derek Lowe says:

      Dang it, I was just talking to some MIT people before writing this post, and it infected me! Fixed, and added a link to the lab.

  2. Chad Irby says:


    1. NJBiologist says:

      You’re five days early….

      1. Michael M says:

        Or 25 days late.

        1. NJBiologist says:

          Yeah, as you can tell, I forgot to switch the calendar page [grinds teeth]….

  3. b says:

    Derek, you may find it interesting to know that Professor Arnold is giving the annual Hill lecture at Duke tomorrow. Always big names on that bill.

  4. Cato says:

    Honest question: what is the work to reward ratio for this kind of enzyme engineering? Obviously some standard enzymes do make it into organic synthesis, but I’m trying to imagine the situation you would go through the effort to engineer this… for example, would you try to utilize this in the scale up of a commercialized drug that requires a tough stereoselective synthesis, etc.. never have done this type of biocatalysis for bench chemistry so curious how easy it is.

    1. anon says:

      that’s only a part of the problem. you also needs TONS of these enzymes to do anything useful in industry. they isolate maybe a few mg of these enzymes and do these reactions at 100-200 mg scale at most.

    2. t says:

      Likely you don’t need to do a ton of engineering to optimize substrate or turnover as, contrary to lock-and-key, most good biocatalysts are fairly promiscuous and with a small handful, you can get pretty wide scope. For heme enzymes, P450s and BM3s, the biggest challenge for industrial application is scalability due to their inherent instability (~2h half life at RT) and low tolerance to co-solvent…hence you must operate at high dilution. That’s a no-go for ton scale production of synthetic intermediates. The best application of this tech currently is around med. chem. and either making cool chiral building blocks or diversifying through late stage functionalizations (all at mg scales). There are companies trying to commercialize this for larger production, so I’m sure progress on addressing stability and dilution issues is being made, but it’s still a long ways from being robust/scalable/general as hydrolases, KREDs, and transaminases. Nonetheless…I’m a huge fan of this work and can’t wait to see where they go next.

      1. Joy says:

        Would immobilization of these enzymes increase their stability?

        1. t says:

          Nope…they don’t like immobilization

          1. DR says:

            Define immobilization. Plenty of enzymes work at surfaces. Adsorbing to a styrene bead, engineering in a click chemistry tag and having a complementary C6 spacer, lodging in a membrane by phobic/philic and protein-protein interactions, all very different.

  5. Peter Kenny says:

    This is great and it’d be interesting to get an idea of C-H bond strengths in these systems. From what I remember, the C-H bonds of cyclopropane are relatively (e.g with respect to cyclohexane) strong and this may be confer metabolic stability. Small strained rings open some options for ‘interpolation’ between the more commonly used alkyls and cycloalkyls. I’m guessing that an amino substituent would not be tolerated?

  6. Stickase says:

    The Arnold lab continues to impress. Although I heard it’s a tough place to work. A friend of a friend got fired as a postdoc immediately when his grant wasn’t funded. Above average need not apply.

    1. Anon says:

      I haven’t heard a single “superstar” whose lab is a fun place to work.

      1. Barry says:


      2. Bla says:

        …in the States. Here in Europe we care about welfare.

        1. Millions of Africans says:

          Sounds great. We’ll be right there!

      3. K G says:

        Grubbs (and many of his former students)? Mark Davis (Chemical Engineering)?

  7. Directed (de)Evolution says:

    Unpopular question: is this really a good scientific training? Arnold herself said about a lot of her work, “if you thought about it, you wouldn’t do it.” Many of the results are completely arbitrary and achievable only by exhaustive screening of mutations. Basically, this work trains students for work that can be done a million times better than robots while simultaneously giving them no intellectual benefit.

    Can directed evolution solve important practical problems? Definitely. Is it a good training for scientists? Not so much IMO.

    1. t says:

      I haven’t really seen a directed evolution program that didn’t rely heavily upon mechanistic insight to at least guide mutation and saturation libraries via modeling, statistical analysis, and chemical insight. I would strongly argue that to be successful in this endeavor, it absolutely requires deep chemical knowledge. It is far from random mutagensis (that’s a very inefficient search algorithm). Often, the first round is random in order to find reactivity hot spots, but from there, you have to understand why that mutation has an effect and make changes and focused mutation libraries to further probe it. There’s also a ton of synthetic creativity that goes into not only choosing reactions and substrates, but understand the “why”. Of course, serendipity and perhaps a spurious observation is the spark that leads down a new pathway of discovery, which is likely what Arnold is alluding to, but one can say that of almost all great discoveries in chemistry.

      1. NMH says:

        Seems like thats the problem with a LOT of academic research these days. My grad advisor does research on the ribosome using computers. Might build up your skills as a computer scientist, but it wont help you with useful scientific skills…

    2. FormerArnoldLabPostdoc says:

      I was there back in the (relatively) early directed evolution days. I found it was a very stimulating, but also very intense, place to work. The lab was about as crowded at 10pm as it was at 11am. We had a fair bit of autonomy within broad parameters as far as hwat we worked on and how we approached it. Also, there was a lot of collective brainstorming about how to optimize libraries so that you actually have a shot at finding something useful, which involved thinking a lot about molecular evolution (which was pretty educational for me, since I came to the lab as a biophysicist).

      So it wasn’t just endless hours of mindless screening (although I remember doing my share of that as well).

  8. NMH says:

    Enough grad students and post-docs in that single (Arnold) lab to fill about all R1 biochem faculty openings west of the Rockies id say. Any they all know the same thing. Does anybody see a problem here?

  9. Andy II says:

    Do you still remember the excitement of “Catalytic Antibody”? That was one of the solutions to address to find a tailor-made catalyst.

  10. F32 Alt F4 says:

    What if enzymes could learn mellatioredox by directed evolved to synthesize secondary metabolites in a diversity oriented synthesis using nothing but visible (!) light, captured CO2, and iridium, enabled by machine learning!!

    1. Anon says:

      Sounds like a Science paper to me.

    2. anon says:

      you forgot to mention gut bacteria

  11. Derek says:


  12. NotAChemist says:

    > “The fact that enzymes can be made to do such chemistry is remarkable”

    You keep pointing out how bad our understanding of biology is. How is this remarkable?

    Nothing personal… I would also note that Prof. Arnold expressed surprise that evolution is a source of innovation. To which I can only say “are you kidding me? Do you not see the monster pile of mysteries that evolution has innovated?”

    1. Derek Lowe says:

      I guess I’m still surprised that it can be harnessed to do things that are so outside its known areas of expertise (!)

  13. milkshake says:

    I had a colleague who did a postdoc on enzyme engineering by mutagenesis at University of Florida. From what he described, it did not look like a particularly promising or practical way of making anything. I could imagine that eventually it could be used maybe for one particular transformation of one particular substrate important to drug or fragrance industry, but not a general method, and the enzyme development is by no means easier or faster than with more conventional catalysts.

    1. anon says:

      Of course not. While chemists like wide open substrate scopes, life generally does not. If you could magically make the enzymes in your body relax their specificity the coroner wouldn’t know where to start.

      Enzymes excel in selectivity (substrate and stereo), rates, and operate under benign reaction conditions. They find plenty of applications in process chemistry (Lipitor and Sitagliptin, for example), and are the primary source for commercial production of natural product APIs and SMs through fermentation. Biocat is complementary to traditional synthetic chemistry. It has a niche, but is not the solution to all problems.

      1. Mol Biologist says:

        Agreed. There is nothing wrong with the satisfaction of a scientific curiosity with the methodical and correct designed research and indeed it may bring “magic bullet”. But IMO there is nothing more intriguing to find the right question in the area where is almost no light.

      2. Anon2 says:

        ” If you could magically make the enzymes in your body relax their specificity the coroner wouldn’t know where to start.”

        LOL. If the enzymes in question were proteases, all they’d find is a puddle.

Comments are closed.